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We narrowed to 527 results for: HA tag

Showing: 261 - 280 of 527 results

  1. Flow Cytometry Readouts: Yes, No, and Everything in Between

    Type
    Blog Post
    ...that for the flow plots discussed in this post, voltage has been adjusted appropriately and compensation ...interpretation. Priyamvada Prathima has a research background in protein mutagenesis and cancer immunotherapies...baseline expression of a protein while the green (top) has an upregulated expression (shift to the right). The...overlaps with the baseline suggesting this sample has a comparable expression to the baseline/negative ...adjusted voltage and compensated your colors. Here are some good practice tips on adjusting voltage: During...changing the voltage for the particular fluorophore prior to recording data. Increasing the voltage will shift...decreasing the voltage will shift it to the left. If your signal is too high, lower the voltage to have your...

  2. CRISPR in the Clinic

    Type
    Blog Post
    ...approaches, has its own unique set of obstacles. Thus far, several targeted therapies have taken advantage of ...There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing field ...CRISPR Therapies Cancer and CAR-T therapy The FDA has already approved CAR-T immunotherapy for cancer treatment...CRISPR Therapeutics. Data shared from these trials has been outstanding, with almost all patients experiencing...desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One treatment...delivery tends to be trafficked to the liver. Thus, it has been used for hATTR therapy, since TTR is primarily...targeting the diseased allele specifically. Early-stage trials showed a high success rate in clearing misfolded...

  3. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    ...diverse bacteria from various environments. It has the advantage of stable genomic integration and is easily...Flp recombinases used to excise markers. It has the advantage that CRISPR efficiency can be controlled by...Cas9-adenosine deaminase fusion Caixia Gao’s lab has developed a new plant base editor from an evolved...String assembly gRNA cloning (STAgR) String assembly gRNA cloning (STAgR), developed by Stefan Stricker...high numbers of gRNAs in one overnight reaction. STAgR is highly customizable as vector backbones, and ...chosen. Read the paper in PLoS One Find the STAgR cloning plasmids Mobile-CRISPRi allows CRISPRi...

  4. Career Coaching for Scientists: Why and Where Do I Find One?

    Type
    Blog Post
    ...to define how you will know that the relationship has been successful – what are the desired outcomes? ... Dora Farkas, founder of Finish Your Thesis (who has also blogged for us) offers a free report on her ...called “Secrets to Success in Graduate School”. She has also launched an online career coaching program that...in Madison, Wisconsin she also works remotely and has experience as a scientific recruiter. Chris Humphrey...of From PhD to Life. She runs great tweet chats, has an outstanding blog and, while her PhD is in History...remotely, will travel for speaking engagements and has many areas of specialty from leadership development... consultation meeting or call for free.  Take advantage of this to get the information you need to choose...

  5. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ... The key advantage of using RNA to reprogram cells is that it’s simple and efficient. It has a high efficiency... field of induced pluripotent stem cells (iPSCs) has been around for 10 years. In that time, scientists... of Myc or other oncogenes after differentiation has been linked to tumor formation in iPSC-derived and...iPSCs compared to other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic...factors driven by one promoter. While lentivirus has similar rates of reprogramming when compared to MMLV-derived...mechanism between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This system is transfected...cell division. A drawback to this approach is it has lower levels of efficiency at generating iPSCs, usually...

  6. Technical Design of a Western Blot

    Type
    Blog Post
    ...agent than BME and has a less… distinctive smell. BME smells like rotting eggs but has a longer shelf life... It doesn’t degrade during long-term storage and has both high binding capacity and high protein retention... specialty low-background PVDF membrane, it also has higher background noise than nitrocellulose membranes... other hand, doesn’t require any pre-wetting and has lower background than PVDF. It is a little quicker...quicker to use, with high binding capacity, though it has lower retention after binding and washes. It’s less...and need to be read fairly soon after the reaction has started. Types of chemiluminescence enzymes The two...darkroom and a developer. Film is highly sensitive but has a limited dynamic range, which means it may take ...

  7. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    ...methods. Each method has its own pluses and minuses, but Golden Gate cloning has been especially useful...of potential clones. Golden Gate assembly has a few advantages over other cloning methods. Exonuclease-...just a few steps. More recently, CRISPR technology has adapted Golden Gate cloning for inserting the appropriate...also be adapted to express multiple gRNAs. Addgene has two Golden Gate-based gRNA assembly methods available...added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning Golden Gate cloning is...destination vector, making multiplexing easy.  Disadvantages of Golden Gate cloning Golden Gate cloning is...

  8. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    ... an essential aspect of biological research that has defined our understanding of biological processes...model organisms is Escherichia coli. The bacterium has been used as a model organism for over 60 years with...cloning and metabolic engineering.  The E. coli genome has an extensive research history and characterization...studies. For example the E. coli biotin ligase, BirA, has been used to identify and study protein-protein interactions...APEX (Rhee et al., 2013). Research using E. coli has the potential to address many areas of microbial ...research with an archive of information and data that has yet to be produced with mice (Iannaccone et al., ..., 2009). While the rat as a model organism has its perks, it is far from perfect. One of the most limiting...

  9. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ...technology has been studied and used in assays for over half a century; the Addgene blog has an excellent...new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium...initially still unstable in eukaryotes, the Jaffrey lab has since developed new RNA-based scaffolds to contain... As biosensors, aptamers hold a few important advantages over their amino acid-based counterparts, antibodies...acceptor FRET know-how with aptamers, aptabeacons (or aptatags) were born. Aptabeacons can be created in many...quenchers are still relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity... Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the nature of GFP fluorescence...

  10. General Transfection

    Type
    Protocol
    ...10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through a 0.22 µm...L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Low serum...L-alanyl-L-glutamine (or stable alternative such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add ...55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots...transfection to determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below...

  11. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...restriction enzyme, you will need to verify that it has be cloned in the correct orientation - this can be... A diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave DNA...

  12. Twenty Years of Addgene Sharing: CRISPR

    Type
    Blog Post
    ...Repair has been viewed 219,289 times, while Validating Your Genome Edit has 76,011 views.  Has all of ...libraries Addgene has distributed (1C), from 2012 to 2023. Addgene’s collection of CRISPR tools has grown steadily...2023, our CRISPR guide has been viewed 1,904,462 times, and the CRISPR 101 eBook has been downloaded over...globe. Our first story focuses on a technology that has changed virtually all of biology…CRISPR.  Since ...own to share. Over the years, Addgene’s repository has grown to include many CRISPR tools.  Distributing... shipped out each year.     Figure 3: Percentage of requests per year for CRISPR plasmids categorized...

  13. Important Considerations When Using AAVs

    Type
    Blog Post
    ...graduate program at the University of Manitoba. Our lab has been utilizing chemogenetics (Designer Receptors ...Although AAV2 can transduce many tissue types, it has low expression in several brain regions where other...serotypes to infect different cell types. In the CNS, it has been shown that there is variable transduction of...from the Original Location - Axonal Transport It has been demonstrated that certain serotypes can undergo... of movement, distance traveled, etc.) (4). This has also been identified with AAV2 (5), AAV6 (6) and ...required for the onset of gene expression. However, it has yet to be determined what gene expression looks like...problem in many experiments, a group of researchers has identified this feature of AAVs as a new tool for...

  14. CRISPR-Cas14: a family of small DNA-targeting enzymes enabling high-fidelity SNP genotyping

    Type
    Blog Post
    ...studied but it is only the tip of the iceberg as it has been predicted that many others are still unknown...enzymes. In contrast to other Cas enzymes, Cas14 has not been found in bacterial genomes but only in the...add-on to a diagnostic toolkit, called DETECTR, which has been created by the authors. This kit uses Cas12 ... Cas systems, Jennifer Doudna’s lab analyzed metagenomic datasets to try to determine whether simpler ...they mined a database of microbial genome and metagenome from the Department of Energy’s Joint Genome ...

  15. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been inserted in...The risk of generating RCL is low but extra care has to be taken especially if you are working with proto-oncogenes...previously used to drive expression from the LTRs) has been removed, Rev (which facilitates nuclear export...expression. In contrast, in the Tet-on system, tTA has been modified (and renamed rtTA) to bind TRE only...viral binding. This method, called spinoculation, has been found very effective for T cells transduction...to infect your cells of interest.   Advantages Disadvantages Can carry large transgenes (>8 kb) Potential... cells, it was obvious that its safety was a disadvantage particularly for clinical development. In addition...

  16. Make a Splash: Notions of Scientific Impact Are Evolving

    Type
    Blog Post
    ... even society. One obvious way to do that is and has been to publish in journals with a high “impact factor...cited articles appearing in lower-prestige journals has risen steadily from 1995 to 2013. Considering citations...online access on scientific impact Partly, the story has to do with online accessibility. It is now about ...among the most cited. And, as scientific discourse has increasingly moved online, surely times have changed...articles, Anurag Acharya and colleagues report, the percentage of citations to articles in non-elite journals...

  17. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    ...also reduced.  Disadvantages Every rose has its thorns and every affinity reagent has its downsides. ...created in lab in situations where a suitable antibody has been identified but a smaller or less immunogenic...using combinatorial libraries, and high specificity has been reported for a number of targets. Due to their...use an alternative affinity reagent?  The main advantage of alternate affinity reagents is their size. ...nanostructures. Diabodies readily co-crystalize, a major advantage over antibodies (Kwon, 2019).  Single-domain antibodies...

  18. Addgene is Stopping Use of X (formerly Twitter)

    Type
    Blog Post
    ...communities and welcome suggestions in the comments.  X has moved significantly away from the functionality and...conversation in the scientific community. Addgene has been considering a gradual move off X as a response...conflict with our values. For this reason, our timeline has accelerated. We significantly reduced posting on ...Bluesky: @addgene.bsky.social; or @addgene on Instagram, TikTok, Facebook, and LinkedIn. We are also exploring...

  19. Cpf1 Update: Comparison to Cas9 and NgAgo

    Type
    Blog Post
    ...regulatory elements. To solve this problem, the Zhang lab has previously used SaCas9 (1053 aa/3.2 kb) with AAV ... these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED ...be the best option for HDR, but this possibility has not been experimentally confirmed. Researchers have...cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than ...2015). PubMed PMID: 26422227.   Potential advantages of Cpf1 Type II CRISPR systems based on Cas9 ...nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18...

  20. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...studying the genetic underpinnings of these processes has been complicated by the lack of precise genetic tools...Simplified in vivo delivery using SPH mice CRISPRa has largely been limited to in vitro settings due to ...can be delivered to the CNS by lentivirus, which has a larger packaging capacity than AAV. Such an approach... or coding regions. Typically, each gene isoform has a distinct transcriptional start site. Because CRISPRa...circumvented by the Yang Lab with the development of a Suntag-p65-HSF1 (SPH) transgenic mouse (Zhou et al., 2018...This mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up to twenty copies...used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA overexpression...
Showing: 261 - 280 of 527 results